In multicellular animals, cell growth, differentiation, and migration are controlled by polypeptide growth factors. These growth factors play a role in both normal development and pathogenesis, including the development of solid tumors. Polypeptide growth factors influence cellular events by binding to cell-surface receptors, many of which are tyrosine kinases. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes, such as cell division, protease production, and cell migration.
Growth factors can be classified into families on the basis of structural similarities. One such family, the PDGF (platelet derived growth factor) family, is characterized by a dimeric structure stabilized by disulfide bonds. This family includes the PDGFs, the placental growth factors (PlGFs), and the vascular endothelial growth factors (VEGFs). The PDGFs are a group of disulfide-bonded, dimeric proteins. Four PDGF polypeptide chains have been identified and named A, B, C, and D chain. The A and B chains forms dimers with themselves and each other, resulting in AA, BB, and AB dimers. See, in general, Ross et al., Cell 46:155–169, 1986 and Hart et al., Biochem. 29:166–172, 1990. Recombinant forms of these proteins, including truncated and substitutional variants, are disclosed in U.S. Pat. Nos. 4,801,542; 4,845,075; 4,849,407; 4,889,919; and 5,895,755. Two additional PDGF polypeptides, designated C and D, have been described. See, WIPO Publication WO 00/34474; WIPO Publication WO 00/66736; Bergsten et al., Nature Cell Biol. 3:512–516, 2001; LaRochelle et al., Nature Cell Biol. 3:517–521, 2001; and Uutela et al., Circulation 103:2242–2247, 2001. PDGF-C is also known as “zvegf3” (WO 00/34474), and PDGF-D is also known as “zvegf4” (WO 00/66736).
PDGF-C and PDGF-D have a multidomain structure that comprises an amino-terminal CUB domain and a carboxyl-terminal growth factor domain joined by an interdomain region of approximately 70 amino acid residues. The growth factor domain of PDGF-D, which comprises approximately residues 250–370 of human PDGF-D (SEQ ID NO:2), is characterized by an arrangement of cysteine residues and beta strands that is characteristic of the “cystine knot” structure of the PDGF family. The CUB domain shows sequence homology to CUB domains in the neuropilins (Takagi et al., Neuron 7:295–307, 1991; Soker et al., Cell 92:735–745, 1998), human bone morphogenetic protein-1 (Wozney et al., Science 242:1528–1534, 1988), porcine seminal plasma protein and bovine acidic seminal fluid protein (Romero et al., Nat. Struct. Biol. 4:783–788, 1997), and Xenopus laevis tolloid-like protein (Lin et al., Dev. Growth Differ. 39:43–51, 1997).
PDGF-C and PDGF-D form homodimeric proteins (PDGF-CC and PDGF-DD) that are proteolytically cleaved to produce the active species, in each case a growth factor domain dimer. The active PDGF-DD protein binds to and activates the α/α, β/β and α/β isoforms of the PDGF receptor. PDGF-DD dimers are mitogenic for a variety of mesenchymal cells (Bergsten et al., ibid.; LaRochelle et al., ibid.). In addition, mice infected with a PDGF-D adenovirus construct showed proliferation of endosteal bone (U.S. patent application Ser. No. 09/540,224).
Production of biologically active, recombinant PDGF-DD has been found to be problematic. See, for example, Bergsten et al., ibid. There is a need in the art for materials and methods for producing recombinant PDGF in economically feasible amounts.